ABSTRACT Serous epithelial ovarian cancer (OvCa) typically presents with wide dissemination of cancer cells within the abdominal cavity and significant tumor burden. OvCa metastases have a high proportion of stroma, which consists primarily of cancer associated fibroblasts (CAFs), a mesenchymal cell type known to promote the invasion and metastasis of tumor cells. However, it is unclear how normal fibroblasts are reprogrammed into CAFs and how CAFs promote tumor growth. To directly address these open questions, we performed proteomic analyses of the primary and metastatic stroma in OvCa patient samples to identify proteins that were differentially expressed. We detected, specifically in the metastases, a conserved stromal signature associated with metastasis that included high stromal expression of Nicotinamide N-Methyltransferase (NNMT). NNMT catalyzes the transfer of a methyl group from S-adenosyl methionine (SAM) to nicotinamide. This depletes cellular SAM stores and leads to global hypomethylation of histones and expression of tumor-promoting genes. In preliminary experiments, we found that NNMT can reprogram normal fibroblasts into CAFs and that NNMT inhibition in CAFs blocks OvCa cancer cell adhesion, proliferation, and in vivo tumor growth. In our system, NNMT expression specifically regulates trimethylation of H3K4 and H3K27. Analysis of NNMT expression in OvCa patients revealed that strong stromal NNMT expression is significantly associated with a poor prognosis. Based on these data, the primary hypothesis underlying this application is that expression of NNMT in normal fibroblasts transforms them to CAFs through metabolically-mediated epigenetic alterations. The proposed experiments will systematically characterize the contribution of NNMT-driven epigenetic and metabolic changes to the transformation of normal fibroblasts to CAFs. In Aim I, we propose to investigate NNMT-driven epigenetic remodeling in the acquisition and maintenance of the CAF phenotype. We will systematically analyze how NNMT regulates the transcriptome and how alteration of epigenetics functionally drives the conversion of normal fibroblasts to CAFs. Since NNMT impinges on multiple metabolic pathways, in Aim II we will systematically assess the NNMT-driven metabolic state and its contribution to CAF differentiation and the promotion of tumor progression. Finally, in Aim III, our group will work with the NIH Center for Advancing Translational Sciences (NCATS) Chemical Genomics Center (NCGC) to discover compounds that inhibit NNMT biochemical activity, using state of the art high-throughput screening with an optimized NNMT biochemical screen of over 300,000 potential inhibitors. Lead compounds will be functionally screened in high- throughput 3D models of the tumor microenvironment at U of C. Successful inhibition of NNMT activity in the tumor stroma could result in a novel and clinically relevant approach to the treatment of metastatic ovarian cancer.